Promoters

ABSTRACT

Isolated DNAs which are DNAs having a base sequence represented by any of SEQ ID NOS:1 to 6 in Sequence Listing or fragments thereof and showing a stationary phase-specific promoter activity in gram positive bacteria.

TECHNICAL FIELD

[0001] The present invention relates to a novel promoter capable ofexpressing a gene product of interest conveniently, at a low cost and ata high level, and a method for producing a protein using the promoter.

BACKGROUND ART

[0002] Expression systems are utilized for producing useful geneproducts by genetic engineering depending on the purposes. In theexpression systems, hosts for which techniques for their cultivationhave been established such as microbial cells (Escherichia coli,Bacillus subtilis, yeast, etc.), animal cells, insect cells and plantcells, and promoters suitable for the hosts are used. Among these, anexpression system in which Escherichia coli as a host and the lacpromoter or a derivative thereof are used is one of the most commonlyused systems because of the operational convenience.

[0003] However, the expression system in which the lac promoter or aderivative thereof is used has a drawback in that it is industriallydisadvantageous because it requires induction of gene expression forexpression of a gene product. For example, induction of gene expressionfrom the lac promoter, the tac promoter or the like requires use of anexpensive reagent, isopropyl-β-D-thiogalactopyranoside (IPTG).Therefore, such a system has a drawback in that it is disadvantageous toperformance on an industrial scale.

[0004] An expression system in which a promoter derived from xyloseoperon and a bacterium of the genus Bacillus as a host are utilized isalso used. However, the system is disadvantageous to performance on anindustrial scale because it requires addition of xylose for theexpression induction.

[0005] Expression vectors that utilize thermoinduction of the phage λpromoter are generally used.

[0006] However, overexpression of recombinant gene products bythermoinduction may be disadvantageous in the following points:

[0007] (a) difficulty in rapidly achieving shifting-up of a temperature;

[0008] (b) increased possibility of forming insoluble inclusion bodiesdue to a higher cultivation temperature; and

[0009] (c) induction of several proteases in Escherichia coli upon heatshock.

[0010]Bacillus subtilis is known to produce and secrete a number ofcatabolic enzymes such as amylases and proteases as a result of astationary phase-specific response. If one could express a gene onlyduring stationary phase after full growth of a host utilizing thestationary phase-specific expression mechanism, the burden on the hostmight be decreased, and an exogenous gene might be efficientlyexpressed. However, no gene expression technique in which such amechanism is utilized has been established.

[0011] Thus, a technique that enables efficient expression withoutartificially inducing gene expression has been desired.

OBJECTS OF INVENTION

[0012] The main object of the present invention is to provide apromoter, a recombinant DNA, a vector for expressing a gene, anexpression vector and a transformed cell that enable expression of agene at a high level in a stationary phase-specific manner withoutartificially inducing the expression of the gene, as well as a methodfor producing a protein which can be carried out conveniently and at alow cost, and a kit for the method.

SUMMARY OF INVENTION

[0013] The present invention is outlined as follows:

[0014] [1] an isolated DNA selected from the group consisting of:

[0015] (a) an isolated DNA having a nucleotide sequence of any one ofSEQ ID NOS:1 to 6 or a fragment thereof which exhibits a promoteractivity in a Gram-positive bacterium in a stationary phase-specificmanner; and

[0016] (b) an isolated DNA hybridizable to the DNA or a fragment thereofof (a) under stringent conditions which exhibits a promoter activity ina Gram-positive bacterium in a stationary phase-specific manner;

[0017] [2] the isolated DNA according to [1], which is capable ofexpressing an exogenous gene in a stationary phase-specific manner inthe absence of an inducer when the DNA is placed upstream of the gene;

[0018] [3] a recombinant DNA in which the DNA defined by [1] and anexogenous gene are placed such that the exogenous gene can be expressed;

[0019] [4] the recombinant DNA according to [3], wherein the exogenousgene is a nucleic acid selected from the group consisting of nucleicacids encoding proteins, nucleic acids encoding antisense RNAs andnucleic acids encoding ribozymes;

[0020] [5] a vector for expressing a gene which contains the DNA definedby [1];

[0021] [6] the vector for expressing a gene according to [5], whereinthe vector is one selected from the group consisting of plasmid vectors,phage vectors and virus vectors;

[0022] [7] an expression vector which contains the recombinant DNAdefined by [3];

[0023] [8] the expression vector according to [7], wherein the vector isone selected from the group consisting of plasmid vectors, phage vectorsand virus vectors;

[0024] [9] a transformed cell which harbors the recombinant DNA definedby [3] or the expression vector defined by [7];

[0025] [10] a method for producing a protein, the method comprising:

[0026] culturing the transformed cell defined by [9]; and

[0027] collecting a protein from the resulting culture; and

[0028] [11] a kit for producing a protein which contains the DNA definedby [1] or the vector for expressing a gene defined by [5].

DETAILED DESCRIPTION OF THE INVENTION

[0029] A DNA derived from Bacillus subtilis DB104 (Gene, 83:215-233(1989)) containing an element that is located upstream of an openreading frame (ORF) for a gene expressed in a stationary phase-specificmanner, and exhibits an promoter activity can be used as the DNA of thepresent invention. The present invention is based on the surprisingfinding by the present inventors that if an exogenous gene (alsoreferred to as a gene of interest) is placed downstream of the DNA, thegene product of interest can be expressed at a high level, i.e., 100 to500 mg per liter of a medium, in the absence of an expression inducer.

[0030] The DNAs of the present invention include DNAs having nucleotidesequences of SEQ ID NOS:1 to 6. The DNA of the present invention ispreferably an isolated DNA that exhibits a promoter activity in astationary phase-specific manner in a Gram-positive bacterium, morepreferably an isolated DNA that exhibits a promoter activity in astationary phase-specific manner in a bacterium of the genus Bacillus orEscherichia coli as described below.

[0031] According to the present invention, “a promoter” comprises aPribnow box, a TATA box or a region similar to the TATA box which islocated about 10 to 30 base pairs upstream from a transcriptioninitiation site (+1) and is responsible for a function of allowing anRNA polymerase to initiate transcription from an exact position. Thepromoter is not necessarily restricted to such a region or surroundingregions, but may comprise, in addition to the region, a region necessaryfor association of a protein other than an RNA polymerase forcontrolling expression. As used herein, the term “a promoter region”refers to a region containing the promoter according to the presentinvention.

[0032] As used herein, “a promoter activity” means that when a constructobtained by placing a gene downstream of a promoter such that the genecan be expressed is introduced into a host, the promoter has an abilityand a function of production of an expression product of the gene insideor outside the host.

[0033] Generally, “a promoter activity” can be measured using a processcomprising:

[0034] (1) a step of connecting a DNA to be subjected to measurement toupstream of a gene encoding a protein that can be readily quantified orobserved (hereinafter also referred to as a reporter gene);

[0035] (2) a step of introducing the resulting construct into a host;

[0036] (3) a step of culturing the resulting transformed cell to expressthe protein; and

[0037] (4) a step of measuring the amount of the expressed protein. Forexample, the presence of “a promoter activity” can be determined byconnecting a sequence that is presumed to have a promoter sequence toupstream of a reporter gene, introducing the construct into a host, andobserving the expression of the gene product inside or outside the host.The observation of expression serves as an index of the promoteractivity of the promoter in the introduced host.

[0038] As used herein, “a stationary phase-specific promoter” refers toa promoter that directs transcription only during stationary phase afterlogarithmic growth phase. “A stationary phase-specific promoter” canexpress a gene placed downstream of the promoter only during stationaryphase without induction using an inducer such as IPTG.

[0039] The DNAs of the present invention include fragments of theisolated DNAs having nucleotide sequences of SEQ ID NOS:1 to 6 as longas the fragments exhibit promoter activities in a stationaryphase-specific manner. “A fragment” can be appropriately selected suchthat it exhibits a promoter activity in a stationary phase-specificmanner. Such a fragment can be selected using the above-mentionedprocess comprising the steps (1) to (4).

[0040] The DNAs of the present invention further include a DNA that hasa nucleotide sequence in which at least one nucleotide, specifically oneor several nucleotides are substituted, deleted, inserted or added inany one of the nucleotide sequences of SEQ ID NOS:1 to 6, and has apromoter activity in a stationary phase-specific manner. In general, theactivity of a DNA having a short sequence may be altered if it has amutation (substitution, deletion, insertion or addition) of at least onenucleotide. However, “a DNA having a mutation” is encompassed by thepresent invention if a promoter activity is observed for the DNA in astationary phase-specific manner using the above-mentioned processcomprising the steps (1) to (4). The mutation may be either a naturallyoccurring or artificially introduced mutation.

[0041] An artificial mutation can be introduced according a conventionalsite-directed mutagenesis method or the like. Examples of thesite-directed mutagenesis methods that can be used include the gappedduplex method which utilizes an amber mutation (Nucleic Acids Research,12:9441-9456 (1984); the Kunkel method in which a host deficient in thedut (dUTPase) and ung (uracil-DNA glycosylase) genes is utilized(Proceedings of the National Academy of Sciences of the USA, 82:488-492(1985)) and a PCR method utilizing an amber mutation (WO 98/02535).

[0042] The present invention also encompasses an isolated DNA that ishybridizable to the complementary strand of the DNA of the presentinvention under stringent conditions or, for example, obtained using anoligonucleotide probe or primer that is designed and chemicallysynthesized according to conventional methods based on the DNA of thepresent invention, and that has a promoter activity in a stationaryphase-specific manner, preferably in a Gram-positive bacterium.

[0043] For example, a DNA for which a promoter activity is observed in astationary phase-specific manner using the above-mentioned processcomprising steps (1) to (4) may be selected as such a DNA. There is nospecific limitation concerning the nucleotide sequence of theoligonucleotide probe as long as the oligonucleotide probe hybridizes tothe DNA or a DNA having a nucleotide sequence complementary to the DNAunder stringent conditions.

[0044] “Stringent conditions” are exemplified by those as described in aliterature such as Sambrook et al., Molecular cloning, A laboratorymanual 2^(nd) edition, 1989, Cold Spring Harbor Laboratory Press. Forexample, the stringent conditions are incubation at a temperature of [Tmof the probe to be used −25° C.] overnight in a solution containing6×SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) 0.5% SDS, 5×Denhardt's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone,0.1% Ficoll 400) and 100 μg/ml of salmon sperm DNA.

[0045] There is also no specific limitation concerning the nucleotidesequence of the primer as long as the primer can anneal to the DNA or aDNA having a nucleotide sequence complementary to the DNA to initiate anextension reaction with a DNA polymerase under conditions used for aconventional PCR.

[0046] Tm of an oligonucleotide probe or primer can be determined, forexample, according to the following equation:

Tm=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)

[0047] wherein N is the chain length of the oligonucleotide probe orprimer; % G+C is the content of guanine and cytosine residues in theoligonucleotide probe or primer.

[0048] If the chain length of the oligonucleotide probe or primer isshorter than 18 nucleotides, Tm can be estimated, for example, as thesum of the product of the number of A+T (adenine and thymine) residuesmultiplied by 2(° C.) and the product of the number of G+C residuesmultiplied by 4(° C.), i.e., [(A+T)×2+(G+C)×4].

[0049] Although it is not intended to limit the present invention, it isdesirable that the chain length of the oligonucleotide probe or primeris preferably 6 nucleotides or more, more preferably 10 nucleotides ormore in order to avoid nonspecific hybridization or annealing.Furthermore, it is desirable that the length is preferably 100nucleotides or less, more preferably 30 nucleotide or less in view ofsynthesis of the oligonucleotide.

[0050] Designing of an oligonucleotide is known to those skilled in theart and can be carried out, for example, with reference to Labo ManualPCR, pp. 13-16, 1996 (Takara Shuzo). Alternatively, a commerciallyavailable software such as OLIGO™ Primer Analysis software (TakaraShuzo) may be used.

[0051] The oligonucleotide can be synthesized according to a knownmethod. For example, it can be synthesized using a DNA synthesizer Model394 (Applied Biosystems) according to the phosphoramidite method.Alternatively, the phosphate triester method, the H-phosphonate method,the thiophosphonate method or the like may be used for the synthesis.

[0052] Using the DNA of the present invention, a recombinant DNA inwhich the DNA and an exogenous gene are placed such that the exogenousgene can be expressed is provided. Such a recombinant DNA is encompassedby the present invention.

[0053] Examples of the exogenous genes include, but are not limited to,nucleic acids encoding proteins (e.g., enzymes, cytokines orantibodies), nucleic acids encoding antisense RNAs and nucleic acidsencoding ribozymes. Examples of the origins of the exogenous genesinclude, but are not limited to, microorganisms (e.g., bacteria, yeasts,actinomycetes, filamentous fungi, ascomycetes and basidiomycetes);plants; insects; and animals. Furthermore, artificially synthesizedgenes may be used depending on the purpose.

[0054] Specifically, examples of the exogenous genes include, but arenot limited to, the interleukin (IL) 1 to 12 genes, the interferon (IFN)α, β and γ genes, the tumor necrosis factor (TNF) gene, thecolony-stimulating factor (CSF) genes, the erythropoietin gene, thetransforming growth factor (TGF)-β gene, the immunoglobulin (Ig) gene,the tissue plasminogen activator (t-PA) gene, the urokinase gene and theWestern firefly luciferase gene.

[0055] As used herein, “a ribozyme” refers to one that cleaves an mRNAfor a specific protein to inhibit the translation of the protein. Aribozyme can be designed on the basis of a sequence of a gene encoding aspecific protein. For example, a hammerhead ribozyme can be preparedusing the method as described in FEBS Letter, 228:228-230 (1988). Theribozymes according to the present invention include any one thatcleaves an mRNA for a specific protein to inhibit the translation of theprotein regardless of the type of the ribozyme (e.g., hammerhead,hairpin or delta).

[0056] The DNA of the present invention exhibits a promoter activitythat enables expression of a gene at a high level even if the geneexpression is not artificially induced. Therefore, it is particularlypreferable for expression of an exogenous gene which is a nucleic acidencoding a protein.

[0057] Furthermore, using the DNA of the present invention, a vector forexpressing a gene that contains the DNA is provided. Such a vector forexpressing a gene is encompassed by the present invention.

[0058] It is possible to express a protein as an example of geneproducts of interest at a level of 100 to 500 mg per liter of a mediumusing the vector for expressing a gene of the present invention becauseit contains the DNA of the present invention. A gene product of interestcan be readily expressed depending on the intended use.

[0059] A plasmid vector, a phage vector or a virus vector, or a vectorfragment consisting of a portion of the vector may be used as a vectorin the vector for expressing a gene of the present invention. The vectoror the vector fragment can be appropriately selected depending on thecell to be use as a host.

[0060] There is no specific limitation concerning the cell that can beused as a host. For example, a Gram-positive bacterium may be used.Examples of the Gram-positive bacteria include bacteria of the genusBacillus for which transformation systems have been established.Specifically, Bacillus subtilis, Bacillus stearothermophilus, Bacilluslicheniformis, Bacillus brevis or Bacillus sp. may be used, although itis not intended to limit the present invention. One obtained bymutagenizing such a bacterium of the genus Bacillus may be used as ahost. Also, Escherichia coli may be used as a host. Transformationsystems for Escherichia coli have been established, Escherichia colicells with various genotypes have been created, and they are readilyavailable. Therefore, Escherichia coli is widely used as a host fortransformation. Specific examples include strains HB101, C600, JM109,DH5α, DH10B, XL-1BlueMRF′ and TOP10F derived from Escherichia coli K-12,although it is not intended to limit the present invention. One obtainedby mutagenizing such an Escherichia coli cell may be used as a host.

[0061] If a bacterium of the genus Bacillus is used as a host, examplesof the vectors include plasmid vectors such as pHY, pUB110 and pE194 aswell as phage vectors such as φ105 and SPβ. If Escherichia coli is usedas a host, examples of the vectors include plasmid vectors such aspUC18, pUC19, pBluescript and pET as well as phage vectors such aslambda phage vectors (e.g., λgt10 and λgt11). The vector for expressinga gene of the present invention which is capable of expressing anexogenous gene in a stationary phase-specific manner can be constructedby appropriately selecting such a vector and incorporating the DNA ofthe present invention into it.

[0062] Techniques as described in Molecular cloning, A laboratory manual2^(nd) edition (supra) or the like can be utilized for the constructionof the vector for expressing a gene of the present invention.Alternatively, the construction may be carried out according to theconstruction procedures as described in Examples below, for example.

[0063] The vector for expressing a gene of the present invention maycontain a terminator (e.g., rrnBT1T), a selectable marker gene and thelike.

[0064] Selectable marker genes include the ampicillin-resistance gene,the kanamycin-resistance gene, the chloramphenicol-resistance gene andthe tetracyclin-resistance gene.

[0065] The vector for expressing a gene of the present invention maycontain the following depending on the intended use of the gene productof interest. For example, it may contain, in order to simplify theprocedure for isolating the gene product of interest, a sequence thatenables expression as a fusion protein with a heterologous protein(e.g., glutathione S-transferase or maltose-binding protein), or a tagsequence that enables expression as a protein to which a histidine tagor the like is added.

[0066] Furthermore, the present invention provides an expression vectorthat contains the above-mentioned recombinant DNA. Such an expressionvector is encompassed by the present invention. The expression vectorsof the present invention include a construct obtained by incorporating agene of interest into the above-mentioned vector for expressing a gene.

[0067] The vector as described above with respect to the vector forexpressing a gene can be used for the expression vector of the presentinvention.

[0068] The expression vector of the present invention can be constructedby (a) incorporating the recombinant DNA into an appropriate vector; (b)incorporating a gene of interest into the vector for expressing a gene;or (c) connecting a gene of interest to the vector fragment forexpressing a gene.

[0069] Using the recombinant DNA or the expression vector of the presentinvention, a transformed cell that harbors the recombinant DNA or theexpression vector can be also provided.

[0070] “The cell that can be used as a host” as described above may beused as a host.

[0071] For example, a recombinant DNA can be introduced into a hostaccording to the method as described in Idenshikougaku Jikken, pp.12-23, Japan Radioisotope Association (ed.) (1991); Virology, 52:456(1973); Molecular and Cellular Biology, 7:2745 (1987); Journal of theNational Cancer Institute, 41:351 (1968); or EMBO Journal, 1:841 (1982).

[0072] For example, an expression vector can be introduced into a hostaccording to the spontaneous competence method (Idenshikougaku Jikken,pp. 12-23, Japan Radioisotope Association (ed.) (1991)); the calciumphosphate method (Molecular and Cellular Biology, 7:2745 (1987)); theelectroporation method (Proc. Natl. Acad. Sci. USA, 81:7161 (1984)); theDEAE-dextran method (Methods in Nucleic Acids Research, pp. 283, Karamet al. (eds.) (1991) CRC Press); or the liposome method (BioTechniques,6:682 (1989)).

[0073] Using the transformed cell of the present invention, a method forproducing a protein comprising culturing the transformed cell, andcollecting a protein from the resulting culture is provided. Such “amethod for producing a protein” is encompassed by the present invention.

[0074] Specifically, a protein can be produced by a method comprising:

[0075] (I) a step of transforming a host cell with:

[0076] (a) a recombinant DNA in which a nucleic acid encoding a proteinis placed downstream of the DNA of the present invention such that thenucleic acid can be expressed; or

[0077] (b) a vector containing the recombinant DNA; and

[0078] (II) a step of culturing the transformed cell obtained in (I) andcollecting the protein from the resulting culture.

[0079] A method for culturing the transformed cell can be appropriatelyselected depending on the cell to be used as a host, the property of theprotein to be expressed and the like.

[0080] The thus obtained protein can be purified by a conventional meansof purifying a protein. Examples of such purification means includesalting out, ion exchange chromatography, hydrophobic chromatography,affinity chromatography and gel filtration chromatography.

[0081] A kit for producing a protein that contains the DNA of thepresent invention or the vector for expressing a gene of the presentinvention can be constructed and used for the method for producing aprotein of the present invention. Using such a kit, a protein can beproduced more conveniently.

EXAMPLES

[0082] The following Examples illustrate the present invention in moredetail, but are not to be construed to limit the scope thereof.

[0083] Relationship between and properties of Bacillus subtilis strainsused according to the present invention are described below.

[0084]B. subtilis Marburg 168: the parent strain of strains generallyused as Bacillus subtilis hosts in recombinant DNA experiments.

[0085]B. subtilis DB104: one of derivatives of B. subtilis Marburg 168which requires histidine. Mutations other than the auxotrophy (nprR2,nprE18, aprED3) are identical to those of UOT1285.

[0086]B. subtilis UOT1285: one of derivatives of B. subtilis Marburg 168which requires tryptophan and lysine. Mutations other than theauxotrophies (nprR2, nprE18, aprED3) are identical to those of DB104.

Example 1 Screening of Genes Expressed in a Stationary Phase-SpecificManner using a Bacillus subtilis DNA Chip

[0087] PCR primers were designed using the DNA sequence information inthe Bacillus subtilis genome database(http://genolist.pasteur.fr/SubtiList/genome.cgi) such that almostfull-length ORFs can be amplified for all the ORFs of Bacillus subtilis.PCRs were carried out using these primers, a genomic DNA from Bacillussubtilis Marburg 168 (Molecular & General Genetics, 152:65-69 (1977))and TaKaRa Ex Taq (Takara Shuzo) or TaKaRa Z-Taq (Takara Shuzo) in a96-well plate.

[0088] The resulting PCR products were purified. The purity and the sizewere checked by agarose gel electrophoresis, and the DNA concentrationwas then calculated by measuring the absorbance for each one.

[0089] The thus obtained DNA fragment solutions were concentrated byisopropanol precipitation. DNA fragments each at a concentration of 1.0μg/ml were immobilized onto a slide glass according to the method asdescribed in WO 00/26404 to prepare a DNA chip.

[0090]Bacillus subtilis UOT1285 (Journal of General Microbiology,135:1335-1345 (1989)) was cultured in 50 ml of 2×SG (1.6% NutrientBroth, 0.05% MgSO₄.7H₂O, 0.2% KCl, 1 mM Ca(NO₃ )₂.4H₂O, 0.1 mMMnCl₂.4H₂O, 0.001 mM FeSO₄.7H₂O, 0.1% glucose) at 37° C. A portion ofthe culture was taken 3, 4 or 5 hours after the initiation ofcultivation, and cells were collected by centrifugation. The thusobtained cells were suspended in TRIZOL Reagent (Gibco BRL). Glass beadswere added thereto. The cells were disrupted using Mini-BeadBeater(Biospec Products). An RNA was recovered by chloroform extraction andisopropanol precipitation according to the protocol attached to TRIZOLReagent. The recovered RNA was treated with RNase-free DNase I (TakaraShuzo), and then recovered by phenol/chloroform extraction followed byethanol precipitation. As a result, about 90-100 μg of RNA was obtained.The RNA was used as an RNA as a template.

[0091] A reverse transcriptase reaction was carried out using 15 μg ofthe RNA as a template prepared as described above and Cy3-dUTP (AmershamPharmacia Biotech) to prepare a Cy3-labeled cDNA probe.

[0092] The DNA chip prepared as described above was subjected toprehybridization in a prehybridization solution (4×SSC, 0.2% SDS, 5×Denhardt's solution, 1 mg/ml of denatured salmon sperm DNA) at roomtemperature for 2 hours, washed in 2×SSC followed by 0.2×SSC, and thendried. Then, hybridization was carried out at 65° C. overnight using theCy3-labeled cDNA probe prepared as described above in a hybridizationsolution which had the same composition as the prehybridization solutionexcept that the concentration of the denatured salmon sperm DNA waschanged to 0.1 mg/ml. After hybridization, the DNA chip was washed in2×SSC containing 0.2% SDS at 55° C. for 30 minutes (twice) and at 65° C.for 5 minutes (once), and in 0.05×SSC at room temperature for 5 minutes,and then dried.

[0093] The hybridized DNA chip was subjected to fluorescence detectionusing a DNA chip analysis apparatus Affymetrix 418 Array Scanner(Affymetrix). The signal intensities obtained by the fluorescencedetection are expressed as color ranks in an image as follows:blue<green<yellow<orange<red<white.

[0094] The thus obtained image data were subjected to measurements andanalyses of signal intensities using an expression data analysissoftware ImaGene (BioDiscovery) according to the instructions attachedto the software. The values of numerically expressed Cy3 signalintensities corrected using the signal for rRNA as an internal standardwere compared, and expression signals for genes at respective growthstages of Bacillus subtilis were determined. As a result, there weregenes for which strong expression signals were observed after 3 hours ofcultivation, genes for which strong expression signals were observedafter 5 hours of cultivation and the like. Thus, it was shown that theexpression patterns of the ORFs were clearly different from each other.

[0095] The growth stage-specificity of expression level was determinedby calculating a ratio of relative expression level by dividing theexpression signal after 4 or 5 hours of cultivation by the expressionsignal after 3 hours of cultivation in order to examine the differencein expression signals in more detail. Results for representative genesare shown in Table 1. TABLE 1 Signal ratio Signal ratio Gene (4 hours/3hours) (5 hours/3 hours) iolJ 4.37 30.5 sigF 1.17 80.3 sipW 0.63 10.9spoIIB 2.21 26.5 spoIIIAH 0.55 10.7 spoIVA 1.30 26.4 yabS 1.93 42.8 ybcO1.15 18.0 ybcP 1.86 34.6 ybcQ 1.89 33.1 ybcS 1.30 27.5 ybcT 1.35 23.2ybdA 1.17 14.3 yjdB 3.11 31.4 yngJ 1.03 6.54 yobH 2.80 4.15 yqxA 0.1031.2 yrzE 2.46 38.4

[0096] As seen from Table 1, there were genes for which expressionlevels were remarkably increased after 5 hours of cultivation, i.e.,during stationary phase.

Example 2 Screening of Genes Expressed in a Stationary Phase-SpecificManner using a Bacillus subtilis Macromembrane

[0097] A digoxigenin (hereinafter referred to as DIG)-labeled cDNA probewas prepared by carrying out a reverse transcriptase reaction using 15μg of the RNA as a template prepared in Example 1 and DIG-11-dUTP (RocheDiagnostics).

[0098] Next, hybridization to Bacillus subtilis DNA array (Eurogentec;hereinafter referred to as a macromembrane; putative ORFs derived fromB. subtilis Marburg 168) was carried out using the DIG-labeled cDNAprobe. For hybridization, prehybridization was carried out in a solutionof DIG Easy Hyb Granules (Roche Diagnostics) at 42° C. for 30 minutes,and hybridization was then carried out at 42° C. overnight. Detectionwas carried out using DIG Wash and Block Buffer and Detection kit (bothfrom Roche Diagnostics).

[0099] The detection results were developed on a photosensitive film.The image was taken into an image analysis apparatus Model GS-700Imaging Densitometer (Bio-Rad) using an image analysis software AdobePhotoshop (Adobe). The signal intensities were measured and analyzedusing an image analysis software MultiAnalyst (Bio-Rad) according to theinstructions attached to the software. Expression signals for genes atrespective growth stages of Bacillus subtilis were measured on the basisof numerically expressed DIG signal intensities. As a result, there weregenes for which strong expression signals were observed after 3 hours ofcultivation, genes for which strong expression signals were observedafter 5 hours of cultivation, and the like. Thus, it was shown that theexpression patterns of the ORFs were clearly different from each other.

[0100] The growth stage-specificity of expression level was determinedby calculating a ratio of relative expression level by dividing theexpression signal after 4 or 5 hours of cultivation by the expressionsignal after 3 hours of cultivation in order to examine the differencein expression signals in more detail. Results for representative genesare shown in Table 2. TABLE 2 Signal ratio Signal ratio Gene (4 hours/3hours) (5 hours/3 hours) acoA 4.42 42.8 acoL 7.17 68.2 sigF 0.79 8.18sipW 8.16 26.3 spoIIB 2.78 11.6 spoIVA 2.15 3.35 yabS 0.80 10.9 ybcO3.45 14.5 ybcP 6.86 31.8 ybcQ 1.41 51.0 ybcS 6.39 53.1 ybcT 3.13 22.2ybdA 1.81 8.95 ybdD 0.20 18.8 yfiA 0.55 5.21 ygaB 0.16 1.65 yjdB 4.1814.4

[0101] As seen from Table 2, there were genes for which expressionlevels were remarkably increased after 5 hours of cultivation, i.e.,during stationary phase.

Example 3 Screening of Stationary Phase-Specific Promoters

[0102] For screening of stationary phase-specific promoters, two roundsof the respective screenings as described in Examples 1 and 2 werecarried out, genes for which expression levels were remarkably increasedafter 5 hours of cultivation, i.e., during stationary phase were totallyjudged on the basis of the results, and the following 23 genes wereselected and used for experiments below: acoA, acoL, iolJ, sigF, sipW,spoIIB, spoIIIAH, spoIVA, yabS, ybcO, ybcP, ybcQ, ybcS, ybcT, ybdA,ybdD, yfiA, ygaB, yjdB, yngJ, yobH, yqxA and yrzE.

[0103] Screening of stationary phase-specific promoters was carried outby connecting an exogenous gene to DNA fragments from the 23 genesexpressed in a stationary phase-specific manner that were presumed tocontain promoter regions for the genes to construct vectors forexpressing the exogenous gene.

[0104] Commercially available enzymes for gel purification and plasmidpurification and kits for gel purification and plasmid purification wereused for constructing the vectors for expressing the gene. Unlessotherwise noted, procedures were carried out according to the methods asdescribed in Molecular cloning, A laboratory manual 2^(nd) edition(Sambrook et al., 1989, Cold Spring Harbor Laboratory Press).

[0105] A total of 46 primers were designed for amplifying about 180-bpDNA fragments containing upstream regions of the 23 genes expressed in astationary phase-specific manner. The regions in the DNA fragments werelocated upstream from the SD sequences for the genes and presumed tocontain promoters. The designing was carried out on the basis of thesequences in the Bacillus subtilis genome database as described inExample 1 (http://genolist.pasteur.fr/SubtiList/genome.cgi). Inaddition, sites for restriction enzymes KpnI and EcoRI were added onboth sides of the respective designed primers in order to simplify theprocedures following amplification.

[0106] The genes from which the DNA fragments presumed to containpromoter regions were to be amplified, the primers used foramplification, and SEQ ID NOS showing the sequences of the primers (inparentheses) are as follows: the acoA gene, primers aAF1 (SEQ ID NO:7)and aAR1 (SEQ ID NO:8); the spoIIB gene, primers spBF1 (SEQ ID NO:9) andspBR1 (SEQ ID NO:10); the ybco gene, primers ybOF1 (SEQ ID NO:11) andybOR1 (SEQ ID NO:12); the yjdB gene, primers yjBF1 (SEQ ID NO:13) andpjBR1 (SEQ ID NO:14); the yngJ gene, primers ynJF1 (SEQ ID NO:15) andynJR1 (SEQ ID NO:16); and the yrzE gene, primers yrEF1 (SEQ ID NO:17)and yrER1 (SEQ ID NO:18).

[0107] A genomic DNA was prepared from Bacillus subtilis DB104 (Gene,83:215-233 (1989)) using ISOPLANT kit (Nippon Gene) according to theinstructions attached to the kit.

[0108] A PCR was carried out using the genomic DNA as a template and theprimers designed as described above as follows: 20 cycles of 94° C. for30 seconds, 50° C. for 1 minute, and 72° C. for 1 minute.

[0109] The 23 DNA fragments presumed to contain promoters which wereamplified as described above were used below.

[0110] A gene for a hyperthormostable protease PFUS derived fromPyrococcus furiosus as described in WO 98/56926 was used as an exogenousgene to be used for screening stationary phase-specific promoters.

[0111]Bacillus subtilis DB104/pSP0124ΔC (FERM BP-6294) is a strain thatharbors a plasmid pSPO124ΔC containing the hyperthermostable proteasePFUS gene as described in WO 98/56926. Bacillus subtilis DB104/pSPO124ΔCwas cultured in 5 ml of LB medium containing 10 μg/ml of kanamycin at37° C. overnight. The plasmid pSPO124ΔC was then prepared from collectedcells using QIAGEN Plasmid Mini kit (Qiagen) according to theinstructions attached to the kit. In this case, cells suspended in thebuffer P1 attached to the kit to which lysozyme was added at aconcentration of 4 mg/ml were treated at 37° C. for 30 minutes.

[0112] A 5448-bp DNA fragment was amplified by a PCR using the plasmidpSPO124ΔC as a template and primers PLF1 (SEQ ID NO:19) and PLR1 (SEQ IDNO:20). The fragment contained the SD sequence and the secretion signalof the aprE gene which encodes subtilisin E from Bacillus subtilis, aswell as the structural gene for the hyperthermostable protease PFUS.This fragment was used as a vector fragment below.

[0113] Procedures are described below with respect to a DNA fragmentcontaining a promoter region for the yngL gene as an example.

[0114] The amplified DNA fragment presumed to contain a promoter regionfor the yngJ gene was digested with restriction enzymes KpnI and EcoRI(both from Takara Shuzo) and purified. The DNA fragment was mixed withand ligated to the vector fragment digested with the restriction enzymesKpnI and EcoRI. The reaction mixture was used to transform Bacillussubtilis DB104. The transformed cells were spread on LB platescontaining 1% skim milk and 10 μg/ml of kanamycin. The plates wereincubated at 37° C. for 16 hours.

[0115] Primers UBF1 (SEQ ID NO:21) and SBPR1 (SEQ ID NO:22) which can beused to amplify the DNA fragment containing the promoter region weredesigned in order to select a clone into which the 180-bp DNA fragmentwas inserted from the resulting kanamycin-resistant transformants. A PCRwas carried out using the combination of these two primers and TaKaRa ExTaq (Takara Shuzo) in a reaction mixture containing 1 mM PMSF asfollows: 20 cycles of 94° C. for 30 seconds, 55° C. for 1 minute, and72° C. for 1 minute.

[0116] Thus, a clone into which the 180-bp DNA fragment was inserted wasselected. A plasmid was prepared from the selected transformant anddesignated as pND20.

[0117] The other 22 DNA fragments were subjected to similar procedures.Then, 18 clones in which the promoter sequences were different from thatin pN20 were obtained among clones for which transformants wereobtained. A total of 19 types of plasmids were prepared from thetransformants.

Example 4 Production of Hyperthermostable Protease Using StationaryPhase-Specific Expression Vectors

[0118] (1) Cultivation of Bacillus subtilis cells transformed withplasmids containing hyperthermostable protease PFUS gene and preparationof crude enzyme solutions

[0119]Bacillus subtilis DB104/pND20 is a strain made by introducing,into Bacillus subtilis DB104, the plasmid pND20 which was prepared inExample 3 and contains the hyperthermostable protease PFUS gene.Bacillus subtilis DB104/pND20 was cultured in 1 ml of TKRBS1 medium (20mg/ml of Polypeptone, 2 mg/ml of yeast extract, 10 mg/ml of meatextract, 40 mg/ml of glucose, 20 μg/ml of FeSO₄.7H₂O, 20 μg/ml ofMnSO₄.5H₂O, and 2 μg/ml of ZnSO₄.7H₂O) containing 10 μg/ml of kanamycinat 37° C. 100 μl of the culture was collected 4, 7, 10 or 13 days afterthe initiation of cultivation. The culture was heated at 95° C. for 30minutes, and then centrifuged to collect a supernatant. The supernatantwas used as a crude enzyme solution.

[0120] Crude enzyme solutions were prepared in a similar manner usingBacillus subtilis DB104 harboring one of the other 18 plasmids.

[0121] (2) Comparison of abilities to produce hyperthermostable protease

[0122] Activities of the hyperthermostable protease PFUS were determinedby spectroscopically measuring p-nitroaniline generated by a hydrolysisreaction with the enzyme using Suc-Ala-Ala-Pro-Phe-p-NA (Sigma) as asubstrate.

[0123] Specifically, an enzyme preparation for which the enzymaticactivity was to be determined was appropriately diluted with 100 mMphosphate buffer (pH 7.0). 50 μl of a solution containingSuc-Ala-Ala-Pro-Phe-p-NA at a concentration of 1 mM in 100 mM phosphatebuffer (pH 7.0) was added to 50 μl of the sample solution. The mixturewas reacted at 95° C. for 30 minutes. The reaction mixture was thencooled on ice to stop the reaction. Absorbance at 405 nm was measured todetermined the amount of generated p-nitroaniline.

[0124] One unit of the enzyme was defined as the amount of the enzymethat generates 1 μmol of p-nitroaniline at 95° C. in 1 minute.

[0125] The amount of the expressed enzyme protein was calculated on thebasis of the determined enzymatic activity assuming that the specificactivity of the hyperthermostable protease PFUS is 9.5 units/mg protein.

[0126] Hyperthermostable protease activities were determined using thecrude enzyme solutions prepared in Example 4-(1) as enzyme preparations.As a result, expression of the hyperthermostable protease PFUS wasobserved using six of the plasmids (pND1, pND6, pND10, pND19, pND20,pND23). Nucleotide sequences of the promoter regions incorporated intothese six plasmids are shown in SEQ ID NOS:1 to 6. Expression levelsobserved using these six plasmids are shown in Table 3. In Table 3, theresults are expressed as relative values defining the expression levelobserved using Bacillus subtilis DB104/pSPO124ΔC as 1. TABLE 3Cultivation time (day) Plasmid 4 7 10 13 pND1 0.10 0.21 0.28 0.29 pND60.01 0.79 1.18 0.86 pND10 1.51 1.53 1.64 1.04 pND19 0.80 0.60 0.61 0.52pND20 1.93 2.45 2.17 2.00 pND23 0.98 0.98 1.01 0.95

[0127] As seen from Table 3, increases in expression levels of thehyperthermostable protease PFUS were observed using pND6 (about1.2-fold), pND10 (about 1.5-fold or more), and pND20 (about 2-fold ormore) as compared with the expression level observed using pSPO124ΔC.Using pND6, almost no expression of the hyperthermostable protease PFUSwas observed on day 4 in the early stage of cultivation. The expressionlevel was greater than that observed using pSPO124ΔC on day 10 in thelater stage of cultivation.

[0128] For plasmids with which expression of the hyperthermostableprotease PFUS was observed, cultivation periods in days which resultedin the maximal expression levels of the hyperthermostable protease PFUS,and the productivities are shown in Table 4 along with the names of thegenes from which the promoters originated. The productivity in Table 4is expressed as the amount in mg per liter of culture. TABLE 4 Gene fromwhich Cultivation promoter Productivity period Plasmid originated (mg)(day) pSPO124ΔC aprE 342 13 pND1 acoA 92.9 13 pND6 spoIIB 310 10 pND10ybcO 514 10 pND19 yjdB 243 10 pND20 yngJ 526 7 pND23 yrzE 279 10

[0129] As seen from Table 4, a greater amount of the hyperthermostableprotease PFUS was produced using pND6, pND10 and pND20 in shorter acultivation period (in days) as compared with the results for pSPO124ΔC.

Example 5 Production of Alkaline Protease, Nitrophenylphosphatase,Pyrrolidone Carboxyl Peptidase and Methionyl Aminopeptidase UsingStationary Phase-Specific Expression Vectors

[0130] (1) Preparation of Vectors

[0131] A region excluding the protease PFUS gene as a reporter gene,i.e., a region containing the promoter region, the SD sequence, thesecretion signal and the vector was amplified by a PCR. In the PCR,primers NDF1 (SEQ ID NO:23) and NDR1 (SEQ ID NO:24) as well as one ofthe plasmids pND1, pND6, pND10, pND19 and pND23 which were constructedin Example 3 above, or pSPO124ΔC (control), as a template were used. Theamplified fragments were digested with restriction enzymes SpeI and MluI(both from Takara Shuzo) and purified. These fragments were used asvector fragments below.

[0132] (2) Preparation of Reporter Genes and Construction of ExpressionVectors

[0133] (i) Alkaline Protease Gene

[0134] A plasmid A2GR7310 which contains an alkaline protease gene froma hyperthermophile Aeropyrum pernix K1 was obtained from NationalInstitute of Technology and Evaluation. A region encoding alkalineprotease was amplified by a PCR using the plasmid as a template as wellas primers AP1F1 (SEQ ID NO:25) and AP1R1 (SEQ ID NO:26). The amplifiedfragment was digested with restriction enzymes SpeI and MluI (both fromTakara Shuzo) and purified. This fragment was used as a reporter genefragment below.

[0135] The nucleotide sequence of the alkaline protease gene from A.pernix which was used as a reporter gene is shown in SEQ ID NO:33.

[0136] Recombinant plasmids (expression vectors) obtained by ligatingthe reporter gene fragment to the vector fragments derived from pND6,pND10 and pSPO124ΔC were designated as pND6A1, pND10A1 and pSPOA1,respectively.

[0137] (ii) Nitrophenylphosphatase Gene

[0138] A plasmid A2GR0030 which contains a nitrophenylphosphatase genefrom a hyperthermophile Aeropyrum pernix K1 was obtained from NationalInstitute of Technology and Evaluation. A region encodingnitrophenylphosphatase was amplified by a PCR using the plasmid as atemplate as well as primers AP7F1 (SEQ ID NO:27) and AP7R1 (SEQ IDNO:28). The amplified fragment was digested with restriction enzymesSpeI and MluI (both from Takara Shuzo) and purified. This fragment wasused as a reporter gene fragment below.

[0139] The nucleotide sequence of the nitrophenylphosphatase gene fromA. pernix which was used as a reporter gene is shown in SEQ ID NO:34.

[0140] Recombinant plasmids (expression vectors) obtained by ligatingthe reporter gene fragment to the vector fragments derived from pND10,pND23 and pSPO124ΔC were designated as pND10A7, pND23A7 and pSPOA7,respectively.

[0141] (iii) Pyrrolidone Carboxyl Peptidase Gene

[0142] A plasmid 2708 which contains a pyrrolidone carboxyl peptidasegene from a hyperthermophile Pyrococcus horikoshii OT3 was obtained fromNational Institute of Technology and Evaluation. A region encodingpyrrolidone carboxyl peptidase was amplified by a PCR using the plasmidas a template as well as primers PH1F1 (SEQ ID NO:29) and PH1R1 (SEQ IDNO:30). The amplified fragment was digested with restriction enzymesSpeI and MluI (both from Takara Shuzo) and purified. This fragment wasused as a reporter gene fragment below.

[0143] The nucleotide sequence of the pyrrolidone carboxyl peptidasegene from P. horikoshii which was used as a reporter gene is shown inSEQ ID NO:35.

[0144] Recombinant plasmids (expression vectors) obtained by ligatingthe reporter gene fragment to the vector fragments derived from pND10,pND19 and pSPO124ΔC were designated as pND10P1, pND19P1 and pSPOP1,respectively.

[0145] (iv) Methionyl Aminopeptidase Gene

[0146] A PCR-amplified fragment PH0628PCR for a methionyl aminopeptidasegene from a hyperthermophile Pyrococcus horikoshii OT3 was obtained fromNational Institute of Technology and Evaluation. A region encodingmethionyl aminopeptidase was amplified by a PCR using the fragment as atemplate as well as primers PH2F1 (SEQ ID NO:31) and PH2R1 (SEQ IDNO:32). The amplified fragment was digested with restriction enzymesSpeI and MluI (both from Takara Shuzo) and purified. This fragment wasused as a reporter gene fragment below.

[0147] The nucleotide sequence of the methionyl aminopeptidase gene fromP. horikoshii which was used as a reporter gene is shown in SEQ IDNO:36.

[0148] Recombinant plasmids (expression vectors) obtained by ligatingthe reporter gene fragment to the vector fragments derived from pND1,pND19 and pSPO124ΔC were designated as pND1P2, pND19P2 and pSPOP2,respectively.

[0149] (3) Preparation of Transformants and Expression of Reporter Genes

[0150] The respective expression vectors constructed in Example 5-(2)were used to transform Bacillus subtilis DB104. The transformed cellswere spread on LB plates containing 1% skim milk and 10 μg/ml ofkanamycin. The plates were incubated at 37° C. for 16 hours.

[0151] A clone into which one of the genes encoding the enzymes wasinserted was selected from the resulting kanamycin-resistanttransformants, and cultured in 1 ml of TKRBS1 medium containing 10 μg/mlof kanamycin at 37° C. A culture collected after 10 days of cultivation(7 days only in case of alkaline protease) was heated at 95° C. for 30minutes, and centrifuged to collect a supernatant. The supernatant wasused as a crude enzyme solution (enzyme preparation).

[0152] (4) Measurements of Activities

[0153] (i) Alkaline Protease

[0154] Alkaline protease activities were measured using gelatin (NacalaiTesque) as a substrate as follows.

[0155] An enzyme preparation for which the enzymatic activity was to bedetermined was appropriately diluted with 50 mM sodium phosphate buffer(pH 7.0). The dilution was mixed with an SDS-PAGE loading buffer. Themixture was allowed to stand at room temperature for 30 minutes orlonger, and then subjected to electrophoresis on 10% polyacrylamide gelcontaining SDS and 0.05% gelatin. After electrophoresis, the gel waswashed in 50 mM sodium phosphate buffer (pH 7.0). The washed gel wasincubated in 50 mM sodium phosphate buffer (pH 7.0) at 95° C. for 3hours. The reaction mixture was then cooled on ice to stop the reaction.The gel was stained with Coomassie Blue. The gel image was converted toan image file using an image analysis software Adobe Photoshop (Adobe)and active signals were numerically expressed using NIH image software.

[0156] (ii) Nitrophenylphosphatase

[0157] Nitrophenylphosphatase Activities were Measured Usingp-nitrophenylphosphate (Sigma) as a Substrate as Follows.

[0158] An enzyme preparation for which the enzymatic activity was to bedetermined was appropriately diluted with 100 mM Tris-HCl buffer (pH7.5) containing 1 mM ZnCl₂. 50 μl of a solution containingp-nitrophenylphosphate at a concentration of 2 mM in 100 mM Tris-HClbuffer (pH 7.5) containing 1 mM ZnCl₂ was added to 50 μl of the samplesolution. The mixture was reacted at 95° C. for 10 minutes. The reactionmixture was then cooled on ice to stop the reaction. The amount ofgenerated free phosphate was determined by measuring fluorescenceemission at 590 nm due to excitation at 544 nm using Piper PhosphateAssay Kit (Molecular Probe).

[0159] (iii) Pyrrolidone Carboxyl Peptidase

[0160] Pyrrolidone carboxyl peptidase activities were measured usingpyroglutamic acid 4-methyl-coumaryl-7-amide (hereinafter referred to asPyr-MCA; Peptide Institute) as a substrate as follows.

[0161] An enzyme preparation for which the enzymatic activity was to bedetermined was appropriately diluted with 50 mM phosphate buffer (pH7.0) containing 10 mM DTT and 1 mM EDTA. 50 μl of a solution containingPyr-MCA at a concentration of 0.2 mM in 50 mM phosphate buffer (pH 7.0)containing 10 mM DTT and 1 mM EDTA was added to 50 μl of the samplesolution. The mixture was reacted at 95° C. for 30 minutes. The reactionmixture was then cooled on ice to stop the reaction. The amount ofgenerated MCA was determined by measuring fluorescence emission at 460nm due to excitation at 355 nm.

[0162] One unit of the enzyme was defined as the amount of the enzymethat generates 1 μmol of MCA at 95° C. in one minute.

[0163] (iv) Methionyl Aminopeptidase

[0164] Methionyl aminopeptidase activities were measured usingMet-Ala-Ser (Bachem) as a substrate as follows.

[0165] An enzyme preparation for which the enzymatic activity was to bedetermined was appropriately diluted with 100 mM potassium phosphatebuffer (pH 7.5) containing 0.5 mM CoCl₂. 45 μl of a solution containingMet-Ala-Ser at a concentration of 1 mM in 100 mM potassium phosphatebuffer (pH 7.5) containing 0.5 MM COCl₂ was added to 5 μl of the samplesolution. The mixture was reacted at 75° C. for 5 minutes. The reactionmixture was then cooled on ice and 10 μl of 100 mM EDTA was addedthereto to stop the reaction. 50 μl of a mixture A (100 mM potassiumphosphate buffer (pH 7.5) containing 0.18 mg/ml of L-amino acid oxidase,50 μg/ml of peroxidase and 0.18 mg/ml of o-dianisidine) was addedthereto. The mixture was reacted at 37° C. for 10 minutes. The reactionmixture was then cooled on ice to stop the reaction. Absorbance at 450nm was measured.

[0166] (5) Comparison of Abilities of Production

[0167] The total enzymatic activities contained in the cultures werecalculated on the basis of the enzymatic activities of the enzymepreparations measured as described in Example 5-(4).

[0168] The expression level observed using an expression vector havingthe promoter for the aprE gene (pSPOA1, pSPOA7, pSPOP1 or pSPOP2) wasdefined as 1. Relative expression levels for the respective expressionvectors are shown in Table 5. TABLE 5 Relative expression Reporter geneVector Promoter level A. pernix pND6A1 spoIIB 6.16 alkaline proteasepND10A1 ybcO 23.8 pSPOA1 aprE 1.00 A. pernix pND10A7 ybcO 2.29nitrophenylphosphatase pND23A7 yrzE 1.68 pSPOA7 aprE 1.00 P. horikoshiipND10P1 ybcO 1.43 pyrrolidone carboxyl peptidase pND19P1 yjdB 3.73pSPOP1 aprE 1.00 P. horikoshii pND1P2 acoA 1.81 methionyl aminopeptidasepND19P2 yjdB 1.98 pSPOP2 aprE 1.00

[0169] Industrial Applicability

[0170] The present invention provides a promoter that enables expressionof a gene at a high level in a stationary phase-specific manner withoutinducing the expression of the gene.

[0171] Sequence Listing Free Text

[0172] SEQ ID NO:1; Promoter region on pND1.

[0173] SEQ ID NO:2; Promoter region on pND6.

[0174] SEQ ID NO:3; Promoter region on pND10.

[0175] SEQ ID NO:4; Promoter region on pND19.

[0176] SEQ ID NO:5; Promoter region on pND20.

[0177] SEQ ID NO:6; Promoter region on pND23.

[0178] SEQ ID NO:7; Primer aAF1 for amplifying promoter sequence of acoAgene.

[0179] SEQ ID NO:8; Primer aAR1 for amplifying promoter sequence of acoAgene.

[0180] SEQ ID NO:9; Primer spBF1 for amplifying promoter sequence ofspoIIB gene.

[0181] SEQ ID NO:10; Primer spBR1 for amplifying promoter sequence ofspoIIB gene.

[0182] SEQ ID NO:11; Primer ybOF1 for amplifying promoter sequence ofybco gene.

[0183] SEQ ID NO:12; Primer ybOR1 for amplifying promoter sequence ofybcO gene.

[0184] SEQ ID NO:13; Primer yjBF1 for amplifying promoter sequence ofyjdB gene.

[0185] SEQ ID NO:14; Primer yjBR1 for amplifying promoter sequence ofyjdB gene.

[0186] SEQ ID NO:15; Primer ynJF1 for amplifying promoter sequence ofyngJ gene.

[0187] SEQ ID NO:16; Primer ynJR1 for amplifying promoter sequence ofyngJ gene.

[0188] SEQ ID NO:17; Primer yrEF1 for amplifying promoter sequence ofyrzE gene.

[0189] SEQ ID NO:18; Primer yrER1 for amplifying promoter sequence ofyrzE gene.

[0190] SEQ ID NO:19; Primer PLF1.

[0191] SEQ ID NO:20; Primer PLR1.

[0192] SEQ ID NO:21; Primer UBF1.

[0193] SEQ ID NO:22; Primer SBPR1.

[0194] SEQ ID NO:23; Primer NDF1.

[0195] SEQ ID NO:24; Primer NDR1.

[0196] SEQ ID NO:25; Primer AP1F1 for amplifying coding region ofalkaline protease gene.

[0197] SEQ ID NO:26; Primer AP1R1 for amplifying coding region ofalkaline protease gene.

[0198] SEQ ID NO:27; Primer AP7F1 for amplifying coding regionof-nitrophenyl phosphatase gene.

[0199] SEQ ID NO:28; Primer AP7R1 for amplifying coding region ofnitrophenyl phosphatase gene.

[0200] SEQ ID NO:29; Primer PH1F1 for amplifying coding region ofpyrrolidone-carboxyl peptidase gene.

[0201] SEQ ID NO:30; Primer PH1R1 for amplifying coding region ofpyrrolidone-carboxyl peptidase gene.

[0202] SEQ ID NO:31; Primer PH2F1 for amplifying coding region ofmethionyl aminopeptidase gene.

[0203] SEQ ID NO:32; Primer PH2R1 for amplifying coding region ofmethionyl aminopeptidase gene.

1 36 1 180 DNA Artificial Sequence A sequence for promoter region onpND1. 1 gtcaaaggcc gggtgatatc cggtcttttt tttgcatgct gtaaaacgagacaaatgaat 60 cagtttgaga caaaacgaga cacacgtctc aaactgtctc caaagtgaagatgagaagac 120 tgattttacg ggctcaaaag actggcacac ttcttgcatt tataatggtgaaccctaaat 180 2 190 DNA Artificial Sequence A sequence for promoterregion on pND6. 2 ccgcagggag cattttagcc tatttatacg gtgactctat catttctttttatatcaaaa 60 tggcattggg ctgactgctg aaaaatttga cgaacgtttt ttggacaaggcgacaaaagt 120 cttgttcttt ttttctttgc ctgtgctaaa gtgtgtagca tgaaaagccgacaagaacgg 180 tcaagcaaat 190 3 180 DNA Artificial Sequence A sequencefor promoter region on pND10. 3 ctgtttttct cgagaggata gcttgtcagcttttctattt ttaaagggtt aaaatattct 60 atttatacta attaatgtaa tttttaggataatatacaaa atccccctta cttcgacaat 120 tgcaatctgg tattatcgta tcgcatgggagctatgtcaa tagactctat gcaaaaattg 180 4 184 DNA Artificial Sequence Asequence for promoter region on pND19. 4 tgctattaac attttaaggatggccattct ctctcagcaa ttttccatca taaatacaaa 60 ctctgtgcag ggcacacaatattcttagct caaatcaatt gatcgttcac atattattaa 120 catttattta caaggaaaataattactttt attgaattgt tatagtgcaa gacaaaaaac 180 ttta 184 5 180 DNAArtificial Sequence A sequence for promoter region on pND20. 5ttcccggctt tatcaaaggg ccaagttatt gcgtttacga tatggatggt atgcgcctac 60tgcatgtatt tgctcatccc gctgatatta tcacataaaa aatgattcag ttttaatttt 120cagacttttc ttgtcaggga atgattatag aactcgccta ataggatgtt acaaagatgt 180 6180 DNA Artificial Sequence A sequence for promoter region on pND23. 6tgaccaatat cacaaaatac aatacgactg tgcgaaacgc aatagtgaga agctcttcca 60ttatgcacct ccaactcatt ataggttgca acaaaatgat caatttatgt aagaaaaacc 120gattgcattt cacaaagctt ttacgtctaa ttcatgggat aagggaatac atttttacaa 180 728 DNA Artificial Sequence A sequence of primer aAF1 for amplifyingpromoter sequence of acoA gene. 7 gggggaattc gtcaaaggcc gggtgata 28 8 28DNA Artificial Sequence A sequence of primer aAR1 for amplifyingpromoter sequence of acoA gene. 8 ggggggtacc atttagggtt caccatta 28 9 28DNA Artificial Sequence A sequence of primer spBF1 for amplifyingpromoter sequence of spoIIB gene. 9 gggggaattc ccgcagggag cattttag 28 1028 DNA Artificial Sequence A sequence of primer spBR1 for amplifyingpromoter sequence of spoIIB gene. 10 ggggggtacc atttgcttga ccgttgtt 2811 28 DNA Artificial Sequence A sequence of primer ybOF1 for amplifyingpromoter sequence of ybcO gene. 11 gggggaattc ctgtttttct cgagagga 28 1228 DNA Artificial Sequence A sequence of primer ybOR1 for amplifyingpromoter sequence of ybcO gene. 12 ggggggtacc caatttttgc atagagtc 28 1328 DNA Artificial Sequence A sequence of primer yjBF1 for amplifyingpromoter sequence of yjdB gene. 13 gggggaattc tgctattaac attttaag 28 1428 DNA Artificial Sequence A sequence of primer yjBR1 for amplifyingpromoter sequence of yjdB gene. 14 ggggggtacc taaagttttt tgtcttgc 28 1528 DNA Artificial Sequence A sequence of primer ynJF1 for amplifyingpromoter sequence of yngJ gene. 15 gggggaattc ttcccggctt tatcaaag 28 1628 DNA Artificial Sequence A sequence of primer ynJR1 for amplifyingpromoter sequence of yngJ gene. 16 ggggggtacc acatctttgt aacatcct 28 1728 DNA Artificial Sequence A sequence of primer yrEF1 for amplifyingpromoter sequence of yrzE gene. 17 gggggaattc tgaccaatat cacaaaat 28 1828 DNA Artificial Sequence A sequence of primer yrER1 for amplifyingpromoter sequence of yrzE gene. 18 ggggggtacc ttgtaaaaat gtattccc 28 1930 DNA Artificial Sequence A sequence of primer PLF1. 19 ggggggtacccaaaaggaga gggggatccg 30 20 29 DNA Artificial Sequence A sequence ofprimer PLR1. 20 gggggaattc ttaggaacgt acagacggc 29 21 20 DNA ArtificialSequence A sequence of primer UBF1. 21 aaaggctttt aagccgtctg 20 22 20DNA Artificial Sequence A sequence of primer SBPR1. 22 cctgcgcagacatgttgctg 20 23 29 DNA Artificial Sequence A sequence of primer NDF1.23 ggtgacgcgt aagctttaat gcggtagtt 29 24 29 DNA Artificial Sequence Asequence of primer NDR1. 24 ggggactagt cctccggcag cctgcgcag 29 25 28 DNAArtificial Sequence A sequence of primer AP1F1 for amplifying codingregion of alkaline protease gene. 25 gaggactagt ggtcgctgta gtaactgg 2826 28 DNA Artificial Sequence A sequence of primer AP1R1 for amplifyingcoding region of alkaline protease gene. 26 ggggacgcgt cagcttgagacggcagtc 28 27 35 DNA Artificial Sequence A sequence of primer AP7F1 foramplifying coding region of nitrophenyl phosphatase gene. 27 gaggactagtgtttgcggat ctagacggcg tgata 35 28 35 DNA Artificial Sequence A sequenceof primer AP7R1 for amplifying coding region of nitrophenyl phosphatasegene. 28 ggggacgcgt cacccccctc tgcagaactc gctga 35 29 28 DNA ArtificialSequence A sequence of primer PH1F1 for amplifying coding region ofpyrrolidone-carboxyl peptidase gene. 29 gaggactagt gaagatctta ttgactgg28 30 30 DNA Artificial Sequence A sequence of primer PH1R1 foramplifying coding region of pyrrolidone-carboxyl peptidase gene. 30ggggacgcgt cacctgagtt gtgatgaatg 30 31 30 DNA Artificial Sequence Asequence of primer PH2F1 for amplifying coding region of methionylaminopeptidase gene. 31 gaggactagt ggatgttgac aagcttattg 30 32 30 DNAArtificial Sequence A sequence of primer PH2R1 for amplifying codingregion of methionyl aminopeptidase gene. 32 ggggacgcgt cattccgttgttacagtcac 30 33 1323 DNA Aeropyrum pernix 33 gtggtcgctg tagtaactggtgtaattcag gtgggaacta agatcgcggc tattgcgatc 60 gcgctgatct tcattctgcctctcttccct gtttatacgg gatcggcggc tggggctagc 120 acggttgtga tagctaagattaatcctgag gagtttaacc ctaaggcggt ggaggctctt 180 cagggcaagg taatatatgttgctgatctg gcccccgttg ctataattag cataccagga 240 aaggctgtag gcctgctctctaaactacct ggtgttgtca gcgtttccga ggacggcgtg 300 gtccaggcta tggccaagccgccgtgggct ggcggcggga ataagtctca gcctgccgag 360 gtcctgcctt ggggtgtcgactatatcgat gccgagctag tatggcccga tggggttacc 420 ggctgggttg acgttaacggtgacggggac ggcgagatag aggttgccgt tattgacact 480 ggtgtcgata aggaccatcccgaccttgca ggcaacattg tctgggggat atctgttttg 540 aacggcagga tatcctccaactaccaggat agaaacggcc acggtacaca cgtaacgggc 600 actgtagccg ccatagacaacgatataggg gtgatagggg ttgcacacag cgtggagatc 660 tacgccgtta aagctctcggtaacgggggt tacggcagct ggagcgacct tataatagct 720 atagaccttg ctgtgaaggggccggacggc gtaattgacg ctgatggaga tggcgtcgtc 780 gctggggatc cagacgatgatgctccagag gttatctcca tgagcctagg tgggagcagc 840 ccaccaccag aactccacgacgttatcaag gcggcgtaca accttggaat aactattgtc 900 gcagcagcgg gtaacgacggggcggacagc ccctcatacc ctgcagccta ccctgaggta 960 atagcggtag gcgctatagacgagaacggc aacgtaccta gctggagcaa tagaaaccct 1020 gaggttgctg cacctggagtgaacatacta agcacctacc ccgacgatac ctatgaggag 1080 ctgagcggca ctagcatggcgactccacac gtgtcaggga ctgtggctct aatacaggct 1140 gccaggctgg ccgctggcctccctctactc cctccgggaa gcgagagtga cactactcca 1200 gacaccgtga ggggcgtactgcatactact gctactgacg cgggagaccc aggctacgat 1260 agcctgtatg gatacggtatcatagacgcc tatgacgccg tgcagactgc cgtctcaagc 1320 tga 1323 34 768 DNAAeropyrum pernix 34 gtgtttgcgg atctagacgg cgtgatatgg cttgggcaggaacctataga ggataaccta 60 gtagtgctta ggactctggc gagcgagggt aggcttgtggttctcactaa taattcgacg 120 cgaagtagga gagtttacgc ggctatgctc gagagggtgggcctcgacat agagcccggg 180 aggatagtaa cgagcgccta cagcgccgcc gtcctgctgaagaaaaagct gggaccatcc 240 accgccctgg ttgtagggga ggaggggctg gttgaagagcttgctgtgga gggccatgta 300 gtggcgagct cgagcgacaa catcgacgtt gatgcggtggtcgtcggcct cgacaggaac 360 ctcacctatg ggaagctggc gagggctgcc tccgcaatacacagcgggag ccttttcgtg 420 gcgacgaacc tcgaccacgc cctaccaacc cccagaggcctcataccagg tgcaggatcc 480 attgtggctc ttctggagaa ggcaacgggg gtcaagcctgcgattgtcgc tgggaagccg 540 tccaggggct tggccgaggt tctagagagc cttttcaagccggtcaggcc cctcgtggtg 600 ggtgatagga tagatactga cgtggagttc gccagggcctggggtgttga ttctcttctc 660 gtgctcactg gcctctacag gggtgtcagc atagaggaggcttctaggaa ggctggggag 720 ggggtgaggg ttgccaggag tctcagcgag ttctgcagaggggggtag 768 35 621 DNA Pyrococcus horikoshii 35 atgaagatct tattgactggctttgagccc tttggaggtg acgataagaa tcccactatg 60 gatatagttg aagctctaagcgaaaggata cctgaggtcg ttggggagat actacccgta 120 tccttcaaga gggctagagaaaagctcctc aaggtacttg acgatgttag gccagatata 180 accataaacc tgggcttggccccaggaaga acgcacatat ccgttgagag agtagccgtg 240 aacatgatag atgcgaggattccggataat gatggagagc aaccgaagga tgaacccata 300 gtcgagggag gacctgcagcttattttgca acaataccca ctagggagat agtcgaagag 360 atgaagaaga acggcattccagcggttctc tcttacacgg ctggaactta tctctgcaat 420 ttcgccatgt atttaaccttacacacatca gctaccaagg gatatcccaa gattgctggc 480 ttcatacacg ttccctacactccggatcaa gtcctggaga aaaagaatac tccgagcatg 540 tctctagatt tagaaataaagggagtggag atagcaataa gggttgctca gagcgcgcta 600 cattcatcac aactcaggta g621 36 888 DNA Pyrococcus horikoshii 36 atggatgttg acaagcttat tgaagccggtaaaatagcta aaaaggttag agaagaagcc 60 gttaaacttg caaagccagg agtttcgctcttggaactgg cagagaaaat tgagagtaga 120 atagttgaac ttggaggtaa gccagcttttccggcaaacc tttccctaaa tgaggtcgct 180 gcccactaca ctccttacaa gggagatcaaactgttctca aagagggaga ctatctaaag 240 atagatttgg gagttcatat agatggatacatagctgata ctgctgtaac cgttagggtt 300 ggaatggatt ttgatgaact tatggaagctgctaaagaag ccctggaaag cgcaatttca 360 gttgcaaggg ccggtgtcga agttaaagaactcgggaaag caatagaaaa tgaaattagg 420 aagagaggct ttaaccccat tgtaaaccttagtgggcata aaatagagag gtacaagctt 480 catgctgggg taagcatccc caatatctacagaccccacg ataactatgt cctccaggaa 540 ggagatgtct ttgcaataga accctttgcaacaacgggtg cggggcaagt tatagaagtt 600 ccccccacgt taatatacat gtacgtcagggatgccccag tcaggatggc ccaagccagg 660 tttctgcttg ctaagataaa gagggagtacaagacacttc cctttgctta taggtggttg 720 caaggagaga tgcccgaagg gcagttaaaactagccttaa gatccctgga gagatccggg 780 gccttgtacg gttaccctgt gctaagggagataaggggag gaatggtcac gcagttcgag 840 catacaataa tagttgaaaa agactccgtgactgtaacaa cggaatga 888

1. An isolated DNA selected from the group consisting of: (a) anisolated DNA having a nucleotide sequence of any one of SEQ ID NOS:1 to6 or a fragment thereof which exhibits a promoter activity in aGram-positive bacterium in a stationary phase-specific manner; and (b)an isolated DNA hybridizable to the DNA or a fragment thereof of (a)under stringent conditions which exhibits a promoter activity in aGram-positive bacterium in a stationary phase-specific manner.
 2. Theisolated DNA according to claim 1, which is capable of expressing anexogenous gene in a stationary phase-specific manner when the DNA isplaced upstream of the gene.
 3. A recombinant DNA in which the DNAdefined by claim 1 and an exogenous gene are placed such that theexogenous gene can be expressed.
 4. The recombinant DNA according toclaim 3, wherein the exogenous gene is a nucleic acid selected from thegroup consisting of nucleic acids encoding proteins, nucleic acidsencoding antisense RNAs and nucleic acids encoding ribozymes.
 5. Avector for expressing a gene which contains the DNA defined by claim 1.6. The vector for expressing a gene according to claim 5, wherein thevector is one selected from the group consisting of plasmid vectors,phage vectors and virus vectors.
 7. An expression vector which containsthe recombinant DNA defined by claim
 3. 8. The expression vectoraccording to claim 7, wherein the vector is one selected from the groupconsisting of plasmid vectors, phage vectors and virus vectors.
 9. Atransformed cell which harbors the recombinant DNA defined by claim 3 orthe expression vector defined by claim
 7. 10. A method for producing aprotein, the method comprising: culturing the transformed cell definedby claim 9; and collecting a protein from the resulting culture.
 11. Akit for producing a protein which contains the DNA defined by claim 1 orthe vector for expressing a gene defined by claim 5.